Hepatitis C virus (HCV) was identified over a decade ago and is now known to be the leading cause of non-A and non-B viral hepatitis (Choo et al., Science (1989) 244:359-362; Armstrong et al., Hepatology (2000) 31:777). HCV infects approximately 3% of the world population, an estimated 200 million people (Cohen, J., Science (1999) 285:26). About 30,000 newly acquired HCV infections occur in the United States annually. Additionally, there is a large incidence of HCV infection in developing countries. Although the immune response is capable of clearing HCV infection, the majority of infections become chronic. Most acute infections remain asymptomatic and liver disease usually occurs only after years of chronic infection.
The viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. HCV has a 9.5 kb positive-sense, single-stranded RNA genome and is a member of the Flaviridae family of viruses. At least six distinct, but related genotypes of HCV, based on phylogenetic analyses, have been identified (Simmonds et al., J. Gen. Virol. (1993) 74:2391-2399). The virus encodes a single polyprotein having about 3000 amino acid residues (Choo et al., Science (1989) 244:359-362; Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455; Han et al., Proc. Natl. Acad. Sci. USA (1991) 88:1711-1715).
In particular, as shown in FIG. 1, several proteins are encoded by the HCV genome. The order and nomenclature of the cleavage products of the HCV polyprotein is as follows: NH2—C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. Another protein (F) has also been identified and results from translational frame-shifting within the C gene. Branch et al., Semin. Liver Dis. (2005) 25:105-117. Initial cleavage of the polyprotein is catalyzed by host proteases which liberate three structural proteins, the N-terminal nucleocapsid protein (termed □core□) and two envelope glycoproteins, gpE1 (also known as E) and gpE2 (also known as E2/NS1), as well as nonstructural (NS) proteins that encode the viral enzymes and other activities. The NS regions are termed NS2, NS3, NS4 and NS5. NS2 is an integral membrane protein with proteolytic activity and, in combination with NS3, cleaves the NS2-NS3 junction. The NS3 protease, along with its NS4a cofactor, serves to process the remaining polyprotein. In these reactions, NS3 liberates an NS3 cofactor (NS4a), two proteins (NS4b and NS5a), and an RNA-dependent RNA polymerase (NS5b). Completion of polyprotein maturation is initiated by autocatalytic cleavage at the NS3-NS4a junction, catalyzed by the NS3 serine protease.
E1 is detected as a 32-35 kDa glycoprotein species and is converted by endoglycosidase H into an approximately 18 kDa species. By contrast, E2 glycoprotein displays a complex pattern upon immunoprecipitation consistent with the generation of multiple species (Spaete et al., Virol. (1992) 188.819-830; Selby et al., J. Virol. (1996) 70:5177-5182; Grakoui et al., J. Virol. (1993) 67:1385-1395; Tomei et al., J. Virol. (1993) 67:4017-4026.). The HCV envelope glycoproteins E1 and E2 form a stable complex that is co-immunoprecipitable (Grakoui et al., J. Virol. (1993) 67:1385-1395; Lanford et al., Virology (1993) 197:225-235; Ralston et al., J. Virol. (1993) 67:6753-6761).
Full-length E1 and E2 are retained within the endoplasmic reticulum of cells and lack complex carbohydrate when expressed stably or in a transient Vaccinia virus system (Spaete et al., Virology (1992) 188:819-830; Ralston et al., J. Virol. (1993) 67:6753-6761). Since the E1 and E2 proteins are normally membrane-bound in these expression systems, secreted truncated forms have been produced in order to facilitate purification of the proteins. See, e.g., U.S. Pat. No. 6,121,020. Additionally, intracellular production of E1E2 in Hela cells has been described. See, e.g., International Publication No. WO 98/50556.
The HCV E1 and E2 glycoproteins are of considerable interest because they have been shown to be protective against viral challenge in primate studies. (Choo et al., Proc. Natl. Acad. Sci. USA (1994) 91:1294-1298; Houghton, M. and Abrignani, S., Nature (2005) 436:961-966). Meunier et al., Proc. Natl. Acad. Sci. USA (2005) 102:4560-4565 used retroviral pseudoparticles displaying intact E1 and E2 glycoproteins and found that viral-neutralizing antibodies raised during HCV-1 infections are also able to neutralize HCV genotypes 4, 5 and 6, but have only limited neutralization against HCV genotypes 2 and 3.
Currently, the only available therapies for HCV are IFN-α and ribavirin. Unfortunately, these agents are effective in less than half the patients treated (Poynard et al., Lancet (1998) 352:1426; McHutchison et al., Engl. J. Med. (1998) 339:1485). Therefore, there is an urgent need for the development of efficacious vaccines to prevent HCV infection, as well as for immunotherapies to be used as an alternative, or in conjunction with existing therapies.
T cell immunity to HCV may determine the outcome of HCV infection and disease (Missale et al., J. Clin. Invest. (1996) 98:706; Cooper et al., Immunity (1999) 10:439; and Lechner et al., J. Exp. Med. (2000) 191:1499). Virus-specific T cell responses have been shown to play an important role in resolving acute HCV infections (Shoukry et al., Ann. Rev. Microbiol. (2004) 58:391-424). One study concluded that individuals displaying predominant Th0/Th1 CD4+ T helper responses resolved their HCV infections, while those with Th2-type responses tended to progress to chronicity (Tsai et al., Hepatology (1997) 25:449-458). In addition, it has been shown that there is an inverse correlation between the frequency of HCV-specific cytotoxic T lymphocytes (CTLs) and viral load (Nelson, et al., J. Immunol. (1997) 158:1473). Control of HCV in chimpanzees has been shown to be associated with a Th1 T cell response (Major et al., J. Virol. (2002) 76:6586-6595). In the chimpanzee model, strong and multispecific CD8+ T cell responses have been associated with spontaneous control of HCV, and the emergence of escape mutants has been associated with the development of viral persistence (Weiner et al., Proc. Natl. Acad. Sci. USA (1995) 92:2755-2759). Therefore, HCV-specific T cell responses appear to play an important role in controlling HCV infection.
Despite extensive advances in the development of pharmaceuticals against certain viruses like HIV, control of acute and chronic HCV infection has had limited success (Hoofnagle and di Bisceglie (1997) N. Engl. J. Med. 336:347-356). As explained above, the generation of a strong cytotoxic T lymphocyte (CTL) response may be important for the control and eradication of HCV infections. Thus, there is a need in the art for effective methods of inducing strong CTL responses against HCV.